Method for purifying fully recombinant AAV particles

Abstract

Provided herein are methods for large-scale purification of recombinant adeno-associated virus (rAAV) particles using chromatography. Further provided herein are pharmaceutical compositions comprising the rAAV particles and prepared by the method.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 378,997, filed October 10, 2022, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Adeno-associated virus (AAV) is a non-enveloped virus belonging to the Parvoviridae family. AAV has a linear single-stranded DNA (ssDNA) genome of approximately 4.7 kilobases (kb) that contains three genes, Rep (replication), Cap (capsid), and aap (assembly). The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for viral genome replication and packaging, while the Cap gene encodes the viral capsid proteins (VP; VP1 / VP2 / VP3) required for capsid formation, target cell binding, and internalization.

[0004] Due to its low genetic complexity, AAV has attracted significant interest in the field of gene therapy. This low genetic complexity facilitates the cloning, packaging and delivery of therapeutic gene expression cassettes to target cells. rAAV, which lacks viral genes and contains a gene expression cassette with a gene of interest, has been shown to be a safe and effective gene therapy vector that can deliver the gene of interest to the target in vivo. rAAV has become the main form of gene therapy, and rAAV-based therapies have received regulatory approval in Europe and the United States.

[0005] rAAV particles can be produced in packaging host cell cultures by the co-expression (for replication and packaging) of helper virus AAV Rep and AAV Cap genes. Typically, the host cell is lysed to release rAAV particles and maximize the yield of the rAAV recovered. However, cell lysates contain various cellular components, such as host cell DNA, host cell proteins, culture medium components, and in some cases also contain helper virus or helper virus plasmid DNA. In addition, not all rAAV particles produced and released from host cells contain genomic DNA (complete rAAV particles) with a gene of interest. A large portion of rAAV particles do not contain DNA (empty rAAV particles) or only contain partial genomes (partially filled rAAV particles). Empty rAAV particles and partially filled rAAV particles are considered to be impurities because they increase the dosage of the total AAV used for effective transduction. Therefore, the rAAV particles collected from culture medium and / or cell lysates should be further purified to be suitable for therapeutic use.

[0006] Currently available methods for rAAV purification are not scalable and / or are not suitable for good manufacturing practices. RAAV particles purified using cesium chloride gradient ultracentrifugation as a purification step have been used in some clinical trials, however, these purification methods are not easily scalable. Other methods (such as heparin-based affinity column chromatography and ion exchange chromatography) have been used for the purification of rAAV. However, unlike density gradient centrifugation, chromatographic methods generally cannot achieve separation of empty capsids from complete capsids.

[0007] Therefore, a scalable purification method that allows the enrichment of complete rAAV capsids from empty and partial capsids is needed, particularly to meet the surging demand for rAAV for rAAV-based therapeutics. Summary of the Invention

[0008] The present disclosure relates to a method for purifying rAAV particles. The method allows purification and enrichment of complete rAAV particles suitable for clinical applications. The present disclosure also provides rAAV particles and pharmaceutical compositions produced by the method.

[0009] In one aspect, the present disclosure provides a method for purifying recombinant adeno-associated virus (rAAV) particles, the method comprising:

[0010] a) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles and complete rAAV particles;

[0011] b) contacting the feed composition with the chromatography medium for a time-on-media under conditions that permit binding of the rAAV particles to the chromatography medium, wherein the time-on-media is at least 0.5 hours;

[0012] c) eluting the rAAV particles from the chromatography medium; and

[0013] d) recovering the purified rAAV particles, thereby enriching for complete rAAV particles.

[0014] In some embodiments, the time on the medium is longer than 2 hours. In some embodiments, the time on the medium is 2 to 24 hours. In some embodiments, the time on the medium is longer than 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0015] In some embodiments, the contacting step comprises holding the rAAV particles bound to the chromatographic medium in a holding buffer for a hold duration, wherein the hold duration is 0.5 hours or longer. In some embodiments, the hold duration is 0.5 to 24 hours. In some embodiments, the hold duration is no more than 3 hours. In some embodiments, the hold duration is 0.5 to 3 hours. In some embodiments, the hold duration is about 3 hours.

[0016] In some embodiments, the pH of the holding buffer is between 9.0 and 11. In some embodiments, the pH of the holding buffer is about pH 10.2.

[0017] In some embodiments, the contacting step comprises loading the rAAV particles onto the chromatographic medium for a loading duration. In some embodiments, the loading duration is greater than 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 18 hours. In some embodiments, the loading duration is between 0.5 and 24 hours.

[0018] In some embodiments, the load duration and the hold duration total 0.5 to 24 hours. In some embodiments, the load duration and the hold duration total at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours.

[0019] In some embodiments, the contacting step comprises washing the rAAV particles bound to the chromatography medium with a wash buffer having a pH of 9.0 to 11 after loading the feed composition onto the chromatography medium but before retaining the rAAV particles bound to the chromatography medium. In some embodiments, the pH of the wash buffer is between pH 9.5 and 10.5. In some embodiments, the pH of the wash buffer is about pH 10.2. In some embodiments, the wash buffer comprises Bis-Tris propane (BTP) or glycine.

[0020] In some embodiments, the pH of the feed composition is between 8.0 and 8.9.

[0021] In some embodiments, the chromatography medium is an anion exchange chromatography medium. In some embodiments, the chromatography medium is an affinity chromatography medium.

[0022] In some embodiments, the elution step is performed using a linear salt gradient. In some embodiments, the linear salt gradient comprises about 0.001 mM NaCl to about 1000 mM NaCl. In some embodiments, the linear salt gradient comprises about 0.001 mM NaCl to about 100 mM NaCl. In some embodiments, the elution step is performed using a step salt gradient. In some embodiments, the step salt gradient comprises about 7 mM NaCl to about 500 mM NaCl. In some embodiments, the step salt gradient comprises about 70 mM NaCl to about 100 mM NaCl.

[0023] In some embodiments, the loading step is performed by flowing the feed composition through the chromatography medium at a flow rate of 0.1 CV / min to 5 CV / min.

[0024] In some embodiments, the chromatographic medium comprises one or more amine functional groups. In some embodiments, the one or more amine functional groups are selected from primary amines, secondary amines, tertiary amines, quaternary amine functional groups, or combinations thereof. In some embodiments, the one or more amine functional groups comprise quaternary amine functional groups. In some embodiments, the one or more amine functional groups are bound to a resin, membrane, and / or nanofiber chromatographic medium. In some embodiments, the chromatographic medium comprises a monolith.

[0025] In some embodiments, the elution step is performed in a buffer at pH 9.5-10.5.

[0026] In some embodiments, the rAAV particles comprise an agent selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAVhu68, AAV.Anc80, and AAV.rh90. Capsid proteins of AAV such as L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and / or AAV.HSC16.

[0027] In some embodiments, the rAAV particle comprises a capsid protein of AAVhu68. In some embodiments, the rAAV particle comprises a capsid protein of AAV1. In some embodiments, the rAAV particle comprises a capsid protein of AAV9.

[0028] In some embodiments, the method further comprises the step of determining the yield of the purified rAAV particles. In some embodiments, the yield of the purified rAAV particles is between 65% and 99%.

[0029] In some embodiments, the method further comprises the step of determining the enrichment of whole rAAV particles in the purified rAAV particles. In some embodiments, at least 80% of the purified rAAV particles are whole rAAV particles. In some embodiments, at least 85% of the purified rAAV particles are whole rAAV particles. In some embodiments, 1% to 40% of the rAAV particles in the feed composition are whole rAAV particles.

[0030] In some embodiments, the rAAV particles in the feed composition further comprise partially filled rAAV particles.

[0031] In some embodiments, the chromatography medium is a pre-packed chromatography monolithic column medium. In some embodiments, the chromatography medium is a rigid, high-flow agarose matrix modified with a dextran surface extender and a strong quaternary ammonium (Q) anion exchanger.

[0032] In some embodiments, the purified rAAV particles have at least 95% of the potency of the rAAV particles in the feed composition.

[0033] In some embodiments, the feed composition comprises poloxamer 188.

[0034] In some embodiments, the method further comprises a prior step of contacting a sample comprising rAAV particles with an affinity chromatography medium, thereby providing the feed composition. In some embodiments, the method further comprises a prior step of preparing the sample comprising rAAV particles by depth filtration, concentration, or diafiltration. In some embodiments, the method further comprises a prior step of preparing the sample comprising rAAV particles by depth filtration, concentration, and diafiltration.

[0035] In another aspect, the present disclosure provides a population of rAAV particles prepared by the methods disclosed herein. In some embodiments, the population of rAAV particles comprises a member selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAVhu68, AAV.Anc80, AAV.Anc8 In some embodiments, the population of rAAV particles comprises a capsid protein of an AAV selected from the group consisting of AAV1, AAV9, and AAVhu68.

[0036] In some embodiments, at least 70% of the rAAV particles in the population are complete rAAV particles. In some embodiments, at least 80% of the rAAV particles in the population are complete rAAV particles. In some embodiments, at least 85% or at least 90% of the rAAV particles in the population are complete rAAV particles.

[0037] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising: a population of rAAV disclosed herein and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description and accompanying drawings, in which:

[0039] Figure 1A-1B Provides low load volume (1 column volume (1CV)) conditions ( Figure 1A ) or high load volume (4 column volumes (4CV)) conditions ( Figure 1B ) 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles containing AAVhu68 capsids purified by AEX.

[0040] Figure 2 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles collected from anion exchange chromatography (AEX) purification (without an on-column hold period) performed under high (3.65E+14 GC / mL resin) and low load (2.44E+14 GC / mL resin) conditions.

[0041] Figure 3 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified from a feed composition containing 20 mM BTP buffer at pH 8.8 and eluted at pH 8.8.

[0042] Figure 4 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles containing AAVhu68 capsids purified from sample dilutions without poloxamer. Exclusion of poloxamer reduced the yield and percentage of complete AAVhu68 particles.

[0043] Figures 5A-5C Provides a short load duration (0.85 hours) ( Figure 5A ) or long load duration (for Figure 5B is 14.57 hours, and for Figure 5C Figure 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified under conditions of 20.54 hours (mAU). The short loading duration was achieved by reducing the loading volume, while the long loading duration was achieved by increasing the loading volume.

[0044] Figure 6 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by a method involving a step of maintaining the feed composition in solution for 24 hours prior to loading onto AEX medium. The feed composition contained rAAV particles suspended in 20 mM BTP buffer at high pH (pH 10.2).

[0045] Figure 7 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by a method involving a step of loading a feed composition onto a prepacked chromatography monolithic column medium at low pH (pH 8.8), followed by a step of maintaining the composition on the column (in the chromatography medium) at high pH (pH 10.2) for 3 hours. The feed composition contains rAAV particles suspended in 20 mM BTP buffer at low pH (pH 8.8).

[0046] Figure 8Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by a method involving maintaining a feed composition in solution at high pH (pH 10.2) for 24 hours prior to loading onto AEX medium ("24 hour hold"), or maintaining the feed composition on a column (in AEX medium) at high pH (pH 10.2) for 24 hours after loading ("on column hold"). The feed composition contains rAAV particles suspended in 20 mM BTP buffer at high pH (pH 10.2).

[0047] Figure 9A and Figure 9B A plot of the % intact rAAV particles (y-axis) as a function of time on medium (x-axis) as measured by SEC-MALS at pH 10.2 is provided. While not every molecule is in contact with the medium for this duration, it is an approximation of the time from the first molecule in contact with the medium until elution begins. Figure 9B Circled in the middle, where loading occurred at low pH, and a 3-hour hold was added. Figure 9B Is from Figure 9A A zoomed-in view of a subset of the data (from 0 to 400 on the x-axis).

[0048] Figure 10 Provided are the 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by AEX after loading the feed composition onto the column for 3 hours. The results are from low loading conditions (3.83E+13).

[0049] Figure 11 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by AEX after loading a feed composition (without a hold step) at high loading pH (pH 10.2). The feed composition contained rAAV particles suspended in 20 mM BTP buffer at high pH (pH 10.2).

[0050] Figure 12 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by AEX after loading a feed composition in glycine buffer at a low loading pH (pH 8.8) followed by holding on the column (in chromatography media) at a high pH (pH 10.2) for a duration of 3 hours. The feed composition contained rAAV particles suspended in 20 mM glycine buffer at a high pH (pH 8.8).

[0051] Figure 13A and Figure 13BProvided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by AEX after loading the feed composition at a low loading pH (pH 8.8) followed by holding on the column (in chromatography media) at a high pH (pH 10.2) for a duration of 24 hours. Figure 13A (Fresh) and Figure 13B The feed composition (cleaned in place (CIP) until storage) contained rAAV particles suspended in 20 mM glycine buffer at low pH (pH 8.8).

[0052] Figure 14 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by AEX after loading a feed composition at pH 8.9 and then holding on the column (in chromatography media) at high pH (pH 10.2) for 3 hours. The loading efficiency was 12.8E+13. The feed composition contained rAAV particles suspended in 20 mM glycine buffer at low pH (pH 8.9).

[0053] Figure 15 Provided are 280 nM and 260 nM UV absorbance (mAU) spectra of rAAV particles purified by AEX after loading a feed composition at a low loading pH (pH 8.8) followed by holding on the column at a high pH (pH 10.2) for a duration of 3 hours. The feed composition contained rAAV particles suspended in 20 mM BTP buffer at low pH (pH 8.8). DETAILED DESCRIPTION

[0054] 5.1. Definitions

[0055] As used herein, the term "recombinant adeno-associated virus particles" or "rAAV particles" refers to nuclease-resistant particles (NRPs) comprising an AAV capsid. The AAV capsid can package a heterologous nucleic acid molecule comprising an AAV 5' and / or 3' inverted terminal repeat sequence therein. In some cases, the AAV capsid does not package a heterologous nucleic acid molecule, forming an empty rAAV capsid. The heterologous nucleic acid molecule can include an expression cassette containing a coding sequence operably linked to an expression control sequence. The coding sequence can encode a therapeutic protein. Alternatively, the expression cassette can include sequences for gene editing, shRNA, miRNA or other therapeutic functions.

[0056] In many cases, rAAV particles are referred to as deoxyribonuclease (DNase) resistant particles (DRP). However, in addition to this endonuclease (DNase), exonucleases can also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded DNA and / or double-stranded DNA as well as RNA. Such steps can contain a single nuclease or a mixture of nucleases for different targets, and can be endonucleases or exonucleases.

[0057] The term "nuclease-resistant" indicates that the AAV capsid has been fully assembled. In the case of complete rAAV particles, the AAV capsid surrounding the expression cassette is designed to deliver the transgene to the host cell and protect these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids that may be present during the production process.

[0058] As used herein, the term "complete rAAV particle" refers to an rAAV particle comprising an AAV capsid encapsulating a heterologous nucleic acid molecule containing AAV 5' and / or 3' inverted terminal repeats. The heterologous nucleic acid molecule is also referred to as a "vector genome". As used herein, the term "empty rAAV particle" refers to an rAAV particle lacking such a heterologous nucleic acid molecule. As used herein, the term "partially filled rAAV particle" refers to an rAAV viral particle containing only partially packaged nucleic acid molecules that are insufficient to achieve expression of a gene product. These empty rAAV particles or partially filled rAAV particles do not have the function of transferring the heterologous nucleic acid molecule (e.g., a gene of interest, a minigene) to a host cell.

[0059] Compositions comprising rAAV particles can be analyzed by UV absorbance at about 260 nm and 280 nm. Since the nucleic acid content of the capsid has a significant impact on the A260 and A280 absorbance data, the A260 / A280 ratio obtained in the absorbance data graph can be used to support the identification of complete AAV particles and empty AAV particles or partially filled AAV particles. Typically, the UV260 detection value of complete rAAV particles containing nucleic acid content in the capsid is higher than 280 nm, while the UV280 detection value of empty particles and partially filled particles is higher than UV260 nm. The A260 / A280 ratio can be obtained by integrating the peak area of the individual substances in the UV absorption (mAU) graph at two wavelengths and dividing the A260 measurement value by the A280 measurement value.

[0060] Compositions comprising rAAV particles can be analyzed and characterized using analytical ultracentrifugation (AUC) or multi-angle static light scattering (SEC-MALS) as described in Brunham et al., Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors. Hum Gene Ther Methods. 2015 Dec;26(6):228-42 and McIntosh et al., Comprehensive characterization and quantification of adeno associated vectors by sizeexclusion chromatography and multi angle light scattering. Sci Rep. 2021 Feb 4;11(1):3012, which are incorporated herein by reference in their entireties.

[0061] As used herein, the term "rAAV particle potency" or "potency" refers to the ability of the rAAV particle to transfer its vector genome into target cells. The potency can be measured by detecting gene expression of the viral genome transferred to the target cells. In some cases, the potency is measured by quantifying the vector genome transferred to the target cells. The potency can be measured in vitro or in vivo.

[0062] As used herein, the term "loading duration" or "loading duration of a feed composition" refers to the time required to load a feed composition onto a chromatography medium. The loading duration of a feed composition can be measured as the time between the initial contact of the feed composition with the chromatography medium and the final application of the feed composition to the chromatography medium. The loading duration can vary depending on the volume of the feed composition, the capacity of the chromatography medium, and the loading rate.

[0063] As used herein, the term "holding duration" or "hold duration" refers to the duration of holding, during which a composition remains in a stable state. The holding is typically performed under static conditions. However, if any changes applied to the composition are consistent and stable, the holding can be performed under dynamic conditions. For example, the "hold duration on the column" can refer to the time that the feed composition remains in contact with the column without any new sample or buffer being applied to the column, or the time that the same sample or buffer is continuously and stably applied to the column.

[0064] "Holding duration in solution" refers to the time that a feed composition remains in solution (eg, holding buffer) in the absence of a chromatography medium, typically prior to loading the feed composition onto a chromatography medium.

[0065] As used herein, the term "column volume" or "CV" refers to the volume within a packed column that is not occupied by the medium. This volume can include the interstitial volume (the volume outside the particles) and the internal porosity (pore volume) of the medium itself. The column volume can be used as a unit. For example, 50 CV or 50 column volumes means 50 times the column volume.

[0066] As used herein, the term "time on medium" refers to the time between the initial contact of rAAV particles with the chromatography medium and the elution of the rAAV particles from the chromatography medium. Although each rAAV particle does not remain on the chromatography medium for this duration, it is determined by the time from the initial loading of the feed composition onto the chromatography medium until the start of elution.

[0067] As used herein, the term "time-on-column" refers to the time between the initial contact of rAAV particles with the chromatography column and the elution of the rAAV particles from the chromatography column.

[0068] 5.2. Methods for Purifying rAAV Particles

[0069] The present disclosure provides a method for purifying recombinant adeno-associated virus (rAAV) particles. The method effectively separates rAAV particles (complete rAAV particles) containing heterologous nucleic acid molecules of AAV 5' and / or 3' inverted terminal repeats containing DNA sequence flanks from rAAV particles lacking the heterologous nucleic acid molecules (empty rAAV particles) or rAAV particles containing partial heterologous nucleic acid molecule DNA (partially filled rAAV particles). In a preferred embodiment, the rAAV composition purified and recovered by the method disclosed herein comprises complete rAAV particles significantly enriched compared to the feed composition.

[0070] The method of purifying rAAV particles comprises applying a feed composition comprising rAAV particles to a chromatography medium under conditions that allow the rAAV particles to bind to the chromatography medium. The rAAV particles bound to the chromatography medium are washed and eluted. To improve performance, the rAAV particles bound to the chromatography medium can be incubated in a holding buffer for at least 0.5 hours before being eluted.

[0071] Thus, in some embodiments, the method comprises:

[0072] a) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles, partially filled rAAV particles, and complete rAAV particles;

[0073] b) loading the feed composition onto the chromatography medium under conditions that permit binding of the rAAV particles to the chromatography medium for a loading duration;

[0074] c) optionally, maintaining the rAAV particles bound to the chromatography medium in a holding buffer for a holding duration, wherein the holding duration is 0.5 hours or longer;

[0075] d) eluting the rAAV particles from the chromatography medium; and

[0076] e) recovering the purified rAAV particles, thereby enriching for complete rAAV particles.

[0077] In some embodiments, the method comprises the step of maintaining the rAAV particles bound to the chromatography medium for a hold duration of at least 0.5 hours.

[0078] In some other embodiments, the method comprises:

[0079] a) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles, partially filled rAAV particles, and complete rAAV particles;

[0080] b) contacting the feed composition with the chromatography medium under conditions that permit binding of the rAAV particles to the chromatography medium for an on-medium time, wherein the on-medium time is at least 0.5 hours;

[0081] c) eluting the rAAV particles from the chromatography medium; and

[0082] d) recovering the purified rAAV particles, thereby enriching for complete rAAV particles.

[0083] In some embodiments, the chromatographic purification method is used in combination with rAAV production and purification methods known in the art. Depending on the order of the additional steps used to generate rAAV particles relative to the chromatographic purification methods disclosed herein, the steps can be referred to as upstream or downstream methods. Various modifications can be made to the upstream and downstream methods.

[0084] 5.2.1. Providing feed composition

[0085] The feed composition is a composition comprising rAAV particles to be purified. The rAAV particles in the feed composition include empty rAAV particles, partially filled rAAV particles, and complete rAAV particles. The feed composition is loaded onto the chromatographic medium.

[0086] In some embodiments, the feed composition comprises a loading buffer that is compatible with the chromatography medium. In some embodiments, the feed composition comprises a loading buffer that is compatible with anion exchange chromatography medium. In some embodiments, the feed composition comprises a loading buffer that is compatible with affinity chromatography medium.

[0087] In some embodiments, the loading buffer comprises bis-tris propane (BTP). In some embodiments, the loading buffer comprises 10-30mM BTP. In some embodiments, the loading buffer comprises 10mM BTP, 15mM BTP, 20mM BTP, 25mM BTP or 30mM BTP. In some embodiments, the loading buffer comprises glycine. In some embodiments, the loading buffer comprises 10-30mM glycine. In some embodiments, the loading buffer comprises 10mM glycine, 15mM glycine, 20mM glycine, 25mM glycine or 30mM glycine.

[0088] In some embodiments, the loading buffer further comprises poloxamer 188. In some embodiments, the loading buffer does not contain poloxamer 188.

[0089] In some embodiments, the loading buffer further comprises NaCl. In some embodiments, the loading buffer further comprises 10-400 mM NaCl. In some embodiments, the loading buffer further comprises 10-200 mM NaCl. In some embodiments, the loading buffer further comprises 10-100 mM NaCl. In some embodiments, the loading buffer further comprises 10-50 mM NaCl.

[0090] In some embodiments, the loading buffer comprises 20 mM BTP and 10 mM NaCl, pH 10.2. In some embodiments, the loading buffer comprises 20 mM glycine and 10 mM NaCl, pH 10.2.

[0091] In some embodiments, the loading buffer provides conditions that allow the rAAV particles to bind to the chromatography medium when applied to the chromatography medium. In some embodiments, the pH of the feed composition is between 9.5 and 10.5. In some embodiments, the pH of the feed composition is between 10 and 10.5. In some embodiments, the pH of the feed composition is about 10.2. In some embodiments, the pH of the feed composition is between 7.0 and 9.5. In some embodiments, the pH of the feed composition is between 7.5 and 9.5. In some embodiments, the pH of the feed composition is between 8.0 and 9.5. In some embodiments, the pH of the feed composition is between 8.0 and 8.9. In some embodiments, the pH of the feed composition is between 8.2 and 8.8. In some embodiments, the pH of the feed composition is 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0092] In some embodiments, less than 50% of the rAAV particles in the feed composition are intact. In some embodiments, less than 40% of the rAAV particles in the feed composition are intact. In some embodiments, less than 30% of the rAAV particles in the feed composition are intact. In some embodiments, less than 20% of the rAAV particles in the feed composition are intact. In some embodiments, less than 10% of the rAAV particles in the feed composition are intact.

[0093] In some embodiments, at least 45% of the rAAV particles in the feed composition are intact. In some embodiments, at least 40% of the rAAV particles in the feed composition are intact. In some embodiments, at least 30% of the rAAV particles in the feed composition are intact. In some embodiments, at least 20% of the rAAV particles in the feed composition are intact. In some embodiments, at least 10% of the rAAV particles in the feed composition are intact. In some embodiments, at least 5% of the rAAV particles in the feed composition are intact. In some embodiments, at least 1% of the rAAV particles in the feed composition are intact.

[0094] In some embodiments, 1% to 40% of the rAAV particles in the feed composition are complete. In some embodiments, 1% to 35% of the rAAV particles in the feed composition are complete. In some embodiments, 1% to 30% of the rAAV particles in the feed composition are complete. In some embodiments, 1% to 25% of the rAAV particles in the feed composition are complete. In some embodiments, 1% to 20% of the rAAV particles in the feed composition are complete.

[0095] 5.2.1.1 Preparation of feed composition

[0096] The feed composition can be prepared using upstream methods known in the art. For example, the feed composition can be generated by rAAV production methods, followed by concentration and / or purification methods.

[0097] In some embodiments, the feed composition is prepared by a method involving (i) rAAV production, (ii) harvest processing and lysis, and (iii) filtration. In some embodiments, the feed composition is prepared by a method involving (i) rAAV production, (ii) harvest processing and lysis, and (iii) depth filtration and filtration. In some embodiments, the feed composition is prepared by a method involving (i) rAAV production, (ii) harvest processing and lysis, and (iii) depth filtration and filtration, and (vi) TFF1 concentration and buffer exchange. In some embodiments, the method further comprises affinity chromatography purification. In some embodiments, the feed composition is prepared by a method involving (i) cell bank thawing, (ii) inoculum expansion, (iii) rAAV production in a production bioreactor, (iv) harvest processing and lysis, (v) depth filtration and filtration, (vi) TFF1 concentration and buffer exchange, and (vii) affinity chromatography. These steps can be used in different orders. Some exemplary methods are described in Figure 1A-1B Available in.

[0098] 5.2.1.1.1 rAAV Production

[0099] Many methods for producing rAAV vectors are known in the art, including, but not limited to, production from cell culture with transient transfection, stable cell line production, and infectious hybrid virus production systems (which include adenovirus-AAV hybrids, herpes virus-AAV hybrids, and baculovirus-AAV hybrids), as described in U.S. Pat. No. 11,098,286, which is incorporated herein in its entirety. rAAV production cultures for producing rAAV viral particles generally require: 1) suitable host cells, including, for example, human-derived cell lines (such as HeLa, A549 or 293 cells), or in the case of baculovirus production systems, insect-derived cell lines (such as SF-9); 2) suitable helper virus functions, which are provided by wild-type or mutant adenovirus (such as temperature-sensitive adenovirus), herpes virus, baculovirus, or nucleic acid constructs that provide helper functions in trans or cis; 3) a functional AAV rep gene, a functional cap gene and gene product; 4) a transgene flanked by AAV·ITR sequences (such as a therapeutic transgene); and 5) suitable culture medium and culture medium components that support rAAV production.

[0100] A variety of suitable cells and cell lines have been described for producing AAV. These cells can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells (including insect cells, yeast cells, and mammalian cells). Particularly desirable host cells are selected from any mammalian species, including but not limited to cells such as A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 cells (which express functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals (including humans, monkeys, mice, rats, rabbits, and hamsters). In certain embodiments, these cells are suspension-adapted cells. In some embodiments, the cell line used for the rAAV particle purification method as described herein is the HEK293 cell line.

[0101] The host cell can be stably transformed with sequences encoding rep and cap and transfected with adenoviral E1, E2a, and E4ORF6 DNA and a construct carrying an expression cassette as described above. Other cell lines that stably express rep and / or cap can be used, such as B-50 (International Patent Application Publication No. WO 99 / 15685) or those described in U.S. Patent No. 5,658,785, which are incorporated herein in their entirety. Another ideal host cell contains minimal adenoviral DNA sufficient to express E4ORF6.

[0102] The components required for AAV production (e.g., adenoviral E1a, E1b, E2a, and / or E4ORF6 gene products, rep or fragments thereof, cap, the expression cassette, and any other required auxiliary functions) can be delivered to the packaging host cell separately or in combination in the form of any genetic element that transfers the carried sequences. Alternatively, one or more components required for culturing the host cell to package the expression cassette into the AAV capsid can be provided to the host cell in trans using appropriate genetic elements.

[0103] Suitable culture media known in the art can be used for the production of rAAV vectors. These culture media include, but are not limited to, culture media produced by Hyclone Laboratories and JRH, including modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), custom formulations (such as those described in U.S. Patent No. 6,566,118 and Sf-900 IISFM medium as described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom culture medium formulations for producing recombinant AAV vectors).

[0104] The rAAV production medium can be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v / v or w / v). Alternatively, as known in the art, rAAV vectors can be produced under serum-free conditions (which may also be referred to as animal-derived product-free culture medium). One of ordinary skill in the art will appreciate that commercial culture media or custom culture media designed to support the production of rAAV vectors may also be supplemented with one or more cell culture components known in the art, including but not limited to glucose, vitamins, amino acids and / or growth factors, in order to increase the titer of rAAV in the production culture.

[0105] rAAV production cultures can be grown under a variety of conditions (over a wide range of temperatures, for varying lengths of time, etc.) appropriate for the particular host cells being utilized. As is known in the art, rAAV production cultures include attachment-dependent cultures, which can be cultured in suitable attachment-dependent containers (e.g., roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors). rAAV vector production cultures can also include suspension-adapted host cells, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including, for example, shake flasks, spinner flasks, stirred tank bioreactors, and disposable systems (e.g., Wave bioreactor systems). In some embodiments, the host cells are grown in a bioreactor (e.g., In some embodiments, host cells are cultured in shake flasks or wave bioreactors for inoculum expansion prior to large-scale culture. In some embodiments, after inoculum expansion, host cells are cultured in a production bioreactor (e.g., Host cells were cultured in a bioreactor system (Pall).

[0106] 5.2.1.2 Harvesting and lysis

[0107] In some embodiments, the rAAV production culture is harvested and the production cell culture is subsequently lysed. The rAAV vector particles of interest can be harvested from the rAAV production culture by lysing the host cells of the production culture or by harvesting the culture medium from the production culture, provided that the cells are cultured under conditions known in the art to release rAAV particles from intact cells into the culture medium, as more fully described in U.S. Patent No. 6,566,118, which is incorporated herein in its entirety. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, ultrasonic treatment, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0108] At harvest, the rAAV production culture may contain, in addition to rAAV particles, one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) culture medium components, including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins.

[0109] In some embodiments, the harvest is treated with a detergent at a slightly alkaline pH (e.g., 0.16% dodecyldimethylamine oxide (LDAO), 2 mM MgCl2, 20 mM Tris, 400 mM NaCl, pH 8.0). In some embodiments, the harvest is treated with a nuclease or a combination of nucleases to digest any contaminating high molecular weight nucleic acids present in the production culture. Such nucleases can target single-stranded DNA, double-stranded DNA, or RNA. Although the examples illustrate the use of deoxyribonucleases (DNases) (e.g., Benzonase or Turbonuclease), other suitable nucleases are known, many of which are commercially available. The nuclease can be a deoxyribonuclease, for example, a DNase, performed under standard conditions known in the art. Digestion. Therefore, a suitable nuclease or combination of nucleases can be selected. In addition, the nuclease(s) selected for this step can be the same or different from the nuclease(s) used during the upstream method or in the subsequent step after harvesting the cell culture. In some embodiments, the harvest is treated with a salt-active nuclease (Salt Active Nuclease High Quality, SanHQ, 25 U / mL).

[0110] 5.2.1.3 rAAV Particle Clarification and Concentration

[0111] In some embodiments, the method of preparing the feed composition further comprises tangential flow filtration (TFF) for concentrating the rAAV particles, heat inactivation of helper virus, rAAV capture by hydrophobic interaction chromatography, buffer exchange by size exclusion chromatography (SEC), and / or filtration. These steps can be used alone, in various combinations, or in different orders.

[0112] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters (including, for example, a DOHC grade Millipore Millistak+HC Pod filter, an A1HC grade Millipore Millistak+HC Pod filter, a 0.2 μm filter Opticap XL10 MilliporeExpress SHC hydrophilic membrane filter, and a Pall Supracap 50 depth filter capsule with a PDK11 grade double-layer media (2-20 μM retention) and a luer-lock connection).

[0113] Clarification can also be achieved by various other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter of 0.2 μm or larger pore size known in the art. Other suitable depth filters (e.g., in the range of about 0.045 μm to about 0.2 μm) or other filtration techniques can also be used. In some embodiments, clarification is performed by 0.2 μm filtration using a Pall EKV 0.2 μm filter. In some embodiments, the clarification step does not involve centrifugation. In some embodiments, the clarification is performed by ultrafiltration or diafiltration.

[0114] 5.2.1.4 Tangential Flow Filtration (TFF) and Buffer Exchange

[0115] In some embodiments, the rAAV composition is concentrated by tangential flow filtration ("TFF"). Large-scale virus concentration using TFF ultrafiltration has been described by R. Paul et al., HUMAN GENE THERAPY, 4:609-615 (1993). TFF concentration of the feed composition enables the chromatography step of the present invention to be performed on a technically manageable volume of feed composition and allows for a more rational determination of the size of the column without the need for a long recirculation time. In some embodiments, the rAAV feed composition is concentrated at least twice to at least ten times. In some embodiments, the feed composition is concentrated at least ten times to at least twenty times. In some embodiments, the feed composition is concentrated at least twenty times to at least fifty times. In some embodiments, the TFF step is performed using a membrane cassette (Pall Omega 100kDa filter) to remove salts and proteins. Those of ordinary skill in the art will also recognize that TFF can also be used in any step of the purification process where it is desired to exchange buffer before performing the next step in the purification process. In some embodiments, tangential flow filtration (TFF) is performed more than once, for example, before and after chromatographic purification.

[0116] 5.2.2. Contacting the Feed Composition with the Chromatographic Medium

[0117] The methods provided herein involve contacting a feed composition comprising rAAV particles with a chromatographic medium under conditions that allow the rAAV particles to bind to the chromatographic medium. The duration of this contacting step is referred to as the "time on medium." The time on medium can be longer than 2 hours. In some embodiments, the time on medium is 2 to 24 hours. In some embodiments, the time on medium is longer than 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In some embodiments, the time on medium is shorter than 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, or 12 hours. In some embodiments, the time on medium is less than 48 hours, 36 hours, or 24 hours.

[0118] In some embodiments, the contacting step comprises loading the rAAV particles onto the chromatographic medium for a loading duration. The loading duration can be longer than 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 18 hours. In some embodiments, the loading duration is 0.5 to 24 hours. The loading duration can be adjusted by varying the loading volume or loading rate.

[0119] In some embodiments, the contacting step comprises maintaining the rAAV particles bound to the chromatographic medium in a holding buffer for a holding duration. In preferred embodiments, the holding duration is 0.5 hours or longer. In some embodiments, the holding buffer is the same as the loading buffer.

[0120] In some embodiments, the load duration and the hold duration total 0.5 to 24 hours. In some embodiments, the load duration and the hold duration total at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours.

[0121] In some embodiments, the contacting step comprises washing the rAAV particles bound to the chromatography medium with a wash buffer having a pH of 9.0 to 11. The wash can be performed after loading the feed composition onto the chromatography medium but before maintaining the rAAV particles bound to the chromatography medium. In some embodiments, the wash can be performed after maintaining the rAAV particles bound to the chromatography medium. In some embodiments, the wash buffer can be the same as the loading buffer and / or the holding buffer.

[0122] 5.2.3. Loading the Feed Composition with Chromatographic Media

[0123] The feed composition can be loaded onto a chromatography medium for purification of rAAV particles and enrichment of complete rAAV particles. After loading, the feed composition can be contacted with the chromatography medium under conditions that allow the rAAV particles to bind to the chromatography medium.

[0124] In some embodiments, the feed composition has been buffer exchanged with column equilibration / loading buffer. In some embodiments, the feed composition is purified using affinity chromatography media. In some embodiments, the feed composition is purified using anion exchange chromatography (AEX). In some embodiments, the feed composition is purified using affinity chromatography and subsequent anion exchange chromatography. In some embodiments, the feed composition is purified using anion exchange chromatography and subsequent affinity chromatography.

[0125] In some embodiments, the method of preparing a feed composition for anion exchange chromatography comprises a step of affinity chromatography. In some embodiments, the method of preparing a feed composition for affinity chromatography comprises a step of anion exchange chromatography.

[0126] The chromatographic media can have a loading capacity in a range of different sizes. In some embodiments, the loading capacity is between 5E+10 and 5E+15 vg / mL of media. In some embodiments, the loading capacity is between 5E+11 and 5E+15 vg / mL of media. In some embodiments, the loading capacity is between 5E+11 and 5E+14 vg / mL of media. In some embodiments, the loading capacity is between 5E+12 and 5E+14 vg / mL of media. In some embodiments, the loading capacity is between 5E+13 and 10E+13 vg / mL of media.

[0127] In some embodiments, the loading requires at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours.

[0128] In some embodiments, the loading requires less than 30 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 6 hours, or less than 3 hours.

[0129] In some embodiments, the loading requires 3-24 hours, 6-24 hours, 12-24 hours, 18-24 hours, or more than 24 hours.

[0130] The loading time can be increased by increasing the loading volume or reducing the flow rate. The loading time can be reduced by reducing the loading volume or increasing the flow rate. In some embodiments, the loading is performed as a continuous flow. In some embodiments, the loading is performed as a discontinuous flow.

[0131] 5.2.3.1 Affinity chromatography

[0132] In some embodiments, the feed composition is loaded onto an affinity chromatography medium having binding specificity for rAAV. In some embodiments, affinity chromatography is performed using an antibody capture affinity chromatography medium. In some embodiments, the affinity chromatography medium contains rAAV-specific antibodies, e.g., antibodies specific for AAV1, AAV9, or AAVhu68, or other immunoglobulin constructs specific for several rAAV serotypes.

[0133] In one embodiment, the chromatographic medium comprises a solid support that is cross-linked poly(styrene-divinylbenzene) having an average particle size of about 50 μm and has rAAV-specific antibodies. An example of such a commercially available affinity resin is POROS® commercially available from Thermo Fischer Scientific. TM A high-performance affinity resin (POROS CaptureSelect AAVX affinity resin). The resin contains a ligand produced using a technology based on single-domain antibody fragments of camelid origin coupled to the resin via carbonyldiimidazole (CDI). The ligand may comprise a single-domain fragment containing three CDRs that form an antigen-binding domain. In some embodiments, the solid support comprises a polymeric matrix material, such as, in particular, agarose, sepharose, or sephadex.

[0134] In some embodiments, the loading is about 2×10 12 GC / mL to about 5×10 14 GC / mL of medium. In some embodiments, the loading is about 2×10 12 GC / mL to about 5×10 13 GC / mL of medium. In some embodiments, the loading is about 2×10 12 GC / mL to about 5×10 12 The range of GC / mL medium is in the range of GC / mL medium.

[0135] In some embodiments, the maximum flow rate is between about 100 cm / hour and about 600 cm / hour (e.g., 350 cm / hour). In some embodiments, the maximum flow rate is between 100 cm / hour and 400 cm / hour. In some embodiments, the maximum flow rate is between 100 cm / hour and 300 cm / hour. In some embodiments, the maximum flow rate is between 100 cm / hour and 200 cm / hour. In some embodiments, the maximum flow rate is between 200 cm / hour and 400 cm / hour. In some embodiments, the maximum flow rate is between 300 cm / hour and 400 cm / hour. In some embodiments, the maximum flow rate is about 150 cm / hour. In some embodiments, the maximum flow rate is about 300 cm / hour. In some embodiments, the maximum flow rate is less than 150 cm / hour. In some embodiments, the maximum flow rate is less than 300 cm / hour.

[0136] In one embodiment, the feed composition containing the rAAV particles (including empty particles, partially filled particles, and full particles) is loaded onto the chromatography medium in a buffer having a high salt concentration (e.g., about 400 nM NaCl to about 650 mM NaCl or other salt(s) of equivalent ionic strength).

[0137] 5.2.3.2 Anion Exchange Chromatography (AEX)

[0138] In some embodiments, the methods provided herein include purification using anion exchange chromatography (AEX). Anion exchange chromatography (AEX) is a form of ion exchange chromatography that separates samples based on their net surface charge. Anion exchange chromatography (AEX) particularly uses positively charged ligands that have an affinity for targets with a net negative surface charge.

[0139] The present invention relates to methods for purifying rAAV particles using various anion exchange chromatography (AEX) media. The interaction between the AEX media and the rAAV particles is affected by several factors, such as the anion exchanger, flow rate, particle size of the resin, binding capacity, etc. The present invention further relates to specific conditions under which the AEX media can be used to effectively separate, purify, or sub-fractionate rAAV particles. For example, specific buffer conditions for purifying rAAV particles using AEX are disclosed.

[0140] In some embodiments, the AEX medium comprises one or more amine functional groups. In some embodiments, the one or more amine functional groups are selected from primary amines, secondary amines, tertiary amines, quaternary amine functional groups, or combinations thereof. In some embodiments, the one or more amine functional groups comprise quaternary amine functional groups. In some embodiments, the one or more amine functional groups are bound to a resin, membrane, and / or nanofiber chromatography medium.

[0141] In some embodiments, the AEX medium comprises a monolith. In some embodiments, the AEX medium is a monolith anion exchange medium. In some embodiments, the AEX medium is a monolith column. In some embodiments, the AEX medium is a pre-packed chromatographic monolithic column medium. In some embodiments, the AEX medium is a CIMQAMonolith column (Sartorius). In some embodiments, the AEX medium comprises a rigid, high-flow agarose matrix modified with a dextran surface extender and a strong quaternary ammonium (Q) anion exchanger.

[0142] Prior to loading, the AEX medium can be equilibrated. In some embodiments, the AEX medium is equilibrated with a loading buffer. In some embodiments, the AEX medium is equilibrated with different buffers in multiple steps.

[0143] In some embodiments, when the feed composition is loaded onto the AEX column, it has a high salt concentration. In one embodiment, the salt concentration of the feed composition is from about 400 mM NaCl to about 650 mM NaCl or equivalent concentrations before being applied to the AEX medium. In one embodiment, the salt concentration of the feed composition is from about 10 mM to about 300 mM NaCl. In one embodiment, the salt concentration of the feed composition is from about 10 mM to about 200 mM NaCl. In one embodiment, the salt concentration of the feed composition is from about 10 mM to about 100 mM NaCl. In one embodiment, the salt concentration of the feed composition is about 10 mM.

[0144] In some embodiments, the feed composition is in load buffer (buffer A). In some embodiments, the buffer comprises 20mM Bis-Tris propane (BTP). In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises 10mM, 15mM, 20mM, 25mM, 30mM or 50mM Bis-Tris propane (BTP). In some embodiments, the buffer comprises 10mM, 15mM, 20mM, 25mM, 30mM or 50mM glycine. In some embodiments, the load buffer comprises 20mM BTP and 10mM NaCl. In some embodiments, the load buffer comprises 20mM glycine and 10mM NaCl.

[0145] In some embodiments, the pH of the feed composition prior to application to the AEX medium is between 9.5 and 10.5. In some embodiments, the pH of the feed composition prior to application to the AEX medium is between 10 and 11. In some feed embodiments, the pH of the composition prior to application to the AEX medium is about 10.2. In some embodiments, the pH of the feed composition prior to application to the AEX medium is between 8 and 9. In some embodiments, the pH of the feed composition prior to application to the AEX medium is between 8.5 and 9.5. In some embodiments, the pH of the feed composition prior to application to the AEX medium is between 8.5 and 9. In some embodiments, the pH of the feed composition prior to application to the AEX medium is about 8.8.

[0146] In some embodiments, the loading is about 2×10 12 GC / mL to about 5×1014 GC / mL of medium. In some embodiments, the loading is about 2×10 12 GC / mL to about 5×10 13 GC / mL of medium. In some embodiments, the loading is about 5×10 12 GC / mL to about 10×10 13 GC / mL of medium. In some embodiments, the loading is about 1×10 13 GC / mL to about 10×10 13 GC / mL of medium. In some embodiments, the loading is about 1×10 13 GC / mL to about 5×10 13 In some embodiments, the loading capacity is about 3.0×10 13 to 3.5×10 13 GC / mL. In some embodiments, the loading is about 2.0×10 14 to 3.5×10 14 GC / mL.

[0147] In some embodiments, the load is carried out at a load rate of 0.1-5.0 column volumes (CV) mL / min AEX medium. In some embodiments, the load is carried out at a load rate of 0.5-3.0CV mL / min AEX medium. In some embodiments, the load is carried out at a load rate of 0.5CV-2.0CV mL / min AEX medium. In some embodiments, the load is carried out at a load rate of 0.5CV-1.5CV mL / min AEX medium. In some embodiments, the load is carried out at a load rate of 0.5CV-1.0CV mL / min AEX medium. In some embodiments, the load is carried out at a load rate of about 0.5CV, 0.6CV, 0.7CV or 0.8CV mL / min AEX medium. In some embodiments, the load is carried out at a load rate of about 1.0CVmL / min AEX medium. In some embodiments, the load time is adjusted by changing the load rate or by changing the load volume.

[0148] 5.2.4. Washing rAAV Particles Bound to Chromatographic Medium

[0149] In some embodiments, the load is followed by a washing step using a wash buffer. The washing step can increase purity or further aid in enriching, depleting, or isolating the rAAV particles. The wash buffer can be a solution having a specific pH range, salts, organic solvents, small molecules, detergents, zwitterions, amino acids, polymers, and any combination thereof. In some embodiments, the washing step is omitted. In this case, the rAAV particles bound to the chromatographic medium are maintained in a holding buffer without being washed with a wash buffer. In some embodiments, the load buffer is used for washing.

[0150] Wash steps for affinity chromatography can be performed with a wash buffer having a higher salt concentration than the loading buffer (eg, in the range of about 750 mM to about 1 M NaCl or equivalent).

[0151] The washing step for anion exchange chromatography can be performed with a wash buffer having a high pH. The pH of the wash buffer can be between 9.0 and 11. In some embodiments, the pH of the wash buffer is between 9.5 and 10.5. In some embodiments, the pH of the wash buffer is about 10.2.

[0152] In some embodiments, the washing is carried out at a washing rate of 0.5-5 column volumes / min of chromatographic medium. In some embodiments, the washing is carried out at a washing rate of about 1 column volume / min of chromatographic medium. In some embodiments, the washing is carried out at a washing rate of 1.0-10.0 column volumes / min of chromatographic medium. In some embodiments, the washing is carried out at a washing rate of 1.0-5.0 column volumes / min of chromatographic medium. In some embodiments, the washing is carried out at a washing rate of 2.0-4.0 column volumes / min of chromatographic medium. In some embodiments, the washing is carried out at a washing rate of 3.0-4.0 column volumes / min of chromatographic medium. In some embodiments, the washing is carried out at a loading rate of about 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5 column volumes / min of chromatographic medium.

[0153] In some embodiments, the wash is performed with 0.5-50 column volumes of the wash buffer. In some embodiments, the wash is performed with 1-40 column volumes of the wash buffer. In some embodiments, the wash is performed with 1-25 column volumes of the wash buffer. In some embodiments, the wash is performed with 5-25 column volumes of the wash buffer. In some embodiments, the wash is performed with 10-20 column volumes of the wash buffer.

[0154] In some embodiments, the washing step is carried out with a wash buffer. In some embodiments, the wash buffer comprises 20mM Bis-Tris propane (BTP). In some embodiments, the wash buffer comprises 10mM, 15mM, 20mM, 25mM, 30mM or 50mM Bis-Tris propane (BTP). In some embodiments, the wash buffer comprises 20mM Bis-Tris propane (BTP) and NaCl. In some embodiments, the wash buffer comprises glycine. In some embodiments, the wash buffer comprises 10mM, 15mM, 20mM, 25mM, 30mM or 50mM glycine. In some embodiments, the wash buffer comprises 20mM glycine and NaCl. In some embodiments, the wash buffer comprises 20mM BTP and 10mM NaCl, and the pH is 10.2. In some embodiments, the wash buffer comprises 20mM glycine and 10mM NaCl, and the pH is 10.2.

[0155] 5.2.5. Maintaining rAAV Particles Bound to Chromatographic Media

[0156] In some embodiments, the method of purifying rAAV particles comprises the step of maintaining the rAAV particles bound to the chromatography medium in a holding buffer for a holding duration of at least 0.5 hours. This holding step can improve purification or further aid in enriching, depleting, or isolating complete rAAV particles.

[0157] In some embodiments, the hold duration is 0.5 hours or longer. In some embodiments, the hold duration is at least 1 hour. In some embodiments, the hold duration is at least 2 hours. In some embodiments, the hold duration is at least 3 hours. In some embodiments, the hold duration is at least 6 hours. In some embodiments, the hold duration is at least 12 hours. In some embodiments, the hold duration is at least 15 hours.

[0158] In some embodiments, the hold duration is less than 24 hours. In some embodiments, the hold duration is less than 20 hours. In some embodiments, the hold duration is less than 18 hours. In some embodiments, the hold duration is less than 15 hours. In some embodiments, the hold duration is less than 12 hours. In some embodiments, the hold duration is less than 6 hours. In some embodiments, the hold duration is less than 3 hours. In some embodiments, the hold duration does not exceed 3 hours.

[0159] In some embodiments, the hold duration is 0.5 to 24 hours. In some embodiments, the hold duration is 1 to 24 hours. In some embodiments, the hold duration is 2 to 24 hours. In some embodiments, the hold duration is 3 to 24 hours. In some embodiments, the hold duration is 3 to 12 hours. In some embodiments, the hold duration is 3 to 6 hours. In some embodiments, the hold duration is 0.5 to 3 hours. In some embodiments, the hold duration is about 3 to 12 hours. In some embodiments, the hold duration is about 3 hours. In some embodiments, the hold duration is about 12 hours. In some embodiments, the hold duration is about 24 hours. In some embodiments, the hold duration is less than 24 hours.

[0160] In some embodiments, the pH of the holding buffer is between 9.0 and 12. In some embodiments, the pH of the holding buffer is between 9.0 and 11. In some embodiments, the pH of the holding buffer is between 9.0 and 10.5. In some embodiments, the pH of the holding buffer is between 9.5 and 10.5. In some embodiments, the pH of the holding buffer is about 10.2. In some embodiments, the pH of the holding buffer is the same as that of the wash buffer. In some embodiments, the holding buffer is the same as that of the wash buffer. In some embodiments, the holding buffer is the same as that of the load buffer. In some embodiments, the holding buffer comprises 20 mM BTP. In some embodiments, the holding buffer comprises 20 mM glycine.

[0161] In some embodiments, the holding is performed under static conditions without applying a sample to the chromatographic medium. In some embodiments, the holding is performed under dynamic conditions with continuous application of a sample or buffer to the chromatographic medium.

[0162] In some embodiments, when the load duration of feed composition is longer than 1 hour, this maintenance step is omitted.In some embodiments, when the load duration of feed composition is longer than 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours, this maintenance step is omitted.In some embodiments, this load duration and this maintenance duration amount to at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours or 23 hours. In some embodiments, the time on the medium of the feed composition is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours or 23 hours. In some embodiments, the time on the medium of the feed composition is less than 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours or 12 hours. In some embodiments, the time on the medium of the feed composition is between 1 hour and 24 hours. In some embodiments, the time on the medium of the feed composition is between 2 hours and 24 hours. In some embodiments, the time on the medium of the feed composition is between 3 hours and 24 hours. In some embodiments, the time on the medium of the feed composition is between 6 hours and 24 hours. In some embodiments, the feed composition has an on-medium time of between 6 hours and 12 hours.

[0163] 5.2.6. Elution of rAAV Particles

[0164] Selective elution can be achieved by changing the salt, phosphate or calcium concentration, changing the pH, changing the temperature, adding organic modifiers, organic solvents, small molecules, detergents, zwitterions, amino acids, polymers, polyols (sucrose, glucose, trehalose, mannose, sorbitol, mannitol, glycerol, etc.), antioxidants (e.g., methionine), EDTA, EGTA, polysorbate 20, polysorbate 80, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol or urea, adding excipients that change the surface tension of the solution, adding excipients that change the polarity of the solution, changing the residence time to take advantage of different desorption rates between complete rAAV particles and empty rAAV particles, or any combination of the above.

[0165] Elution can be achieved with a variety of elution buffers having different properties (e.g., pH, salt, organic solvents, small molecules, detergents, zwitterions, amino acids, polymers, temperature, and any combination thereof). Multiple eluted fractions can be collected, wherein the rAAV particles collected in each fraction have different properties. For example, the rAAV particles collected in one fraction have a higher purity, a smaller or larger average size, a preferred composition, etc., compared to the rAAV particles in other fractions. Such elution buffers having different properties can be applied as a continuous flow while collecting multiple eluted fractions.

[0166] In some embodiments, the elution step is performed using a linear salt gradient. In some embodiments, the linear salt gradient comprises about 0.001 mM NaCl to about 1000 mM NaCl. In some embodiments, the linear salt gradient comprises about 0.001 mM NaCl to about 100 mM NaCl. In some embodiments, the linear salt gradient comprises about 0.01 mM NaCl to about 100 mM NaCl. In some embodiments, the linear salt gradient comprises about 1 mM NaCl to about 100 mM NaCl.

[0167] In some embodiments, the elution step is performed using a step salt gradient. In some embodiments, the step salt gradient comprises about 7 mM NaCl to about 500 mM NaCl. In some embodiments, the step salt gradient comprises about 70 mM NaCl to about 500 mM NaCl. In some embodiments, the step salt gradient comprises about 70 mM NaCl to about 100 mM NaCl.

[0168] 5.2.7. Recovery of Purified rAAV Particles

[0169] The method of purifying rAAV particles includes the step of recovering or collecting rAAV particles from the elution. The purified rAAV particles can be in an eluate from the elution. The eluate can be analyzed to determine the purity of the purified rAAV particles (e.g., the percentage [%] of complete rAAV particles).

[0170] Thus, the method can further comprise the step of analyzing the purified rAAV particles. In some embodiments, the method comprises the step of determining the yield of the purified rAAV particles. In some embodiments, the method comprises the step of determining the yield of complete rAAV particles. The yield can be determined as the ratio between the total purified rAAV particles in the eluate and the total rAAV particles in the feed composition.

[0171] In some embodiments, the yield is at least 50%. In some embodiments, the yield is at least 60%, 70%, 80%, 90%, or 95%. In some embodiments, the yield is between 65% and 99%. In some embodiments, the yield is between 70% and 99%. In some embodiments, the yield is between 75% and 99%. In some embodiments, the yield is between 80% and 99%. In some embodiments, the yield is between 80% and 95%.

[0172] In some embodiments, the method comprises the step of determining the enrichment of whole rAAV particles in the purified or recovered rAAV particles. In some embodiments, the method comprises the step of determining the enrichment of whole rAAV particles in the purified rAAV particles compared to the whole rAAV particles in the feed composition. In some embodiments, the method comprises the step of determining the percentage (%) of whole rAAV particles in the purified rAAV particles.

[0173] In some embodiments, at least 60% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 65% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 70% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 75% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 80% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 85% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 90% of the purified rAAV particles are complete rAAV particles. In some embodiments, at least 95% of the purified rAAV particles are complete rAAV particles.

[0174] In some embodiments, the purified rAAV particles have at least 99% of the potency of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 98% of the potency of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 95% of the potency of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 90% of the potency of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 85% of the potency of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 80% of the potency of the rAAV particles in the feed composition. In some embodiments, the purified rAAV particles have at least 75% of the potency of the rAAV particles in the feed composition.

[0175] 5.3. Population of Purified rAAV Particles

[0176] In another aspect, the present disclosure provides a population of rAAV particles purified using the methods disclosed herein. In some embodiments, the population comprises enriched whole rAAV particles.

[0177] In some embodiments, at least 95% of the population is complete rAAV particles. In some embodiments, at least 90% of the population is complete rAAV particles. In some embodiments, at least 85% of the population is complete rAAV particles. In some embodiments, at least 80% of the population is complete rAAV particles. In some embodiments, at least 75% of the population is complete rAAV particles. In some embodiments, at least 70% of the population is complete rAAV particles. In some embodiments, at least 99%, 98%, 97%, 98%, 96%, or 95% of the population is complete rAAV particles.

[0178] In some embodiments, less than 5% of the population are empty rAAV particles. In some embodiments, less than 10% of the population are empty rAAV particles. In some embodiments, less than 15% of the population are empty rAAV particles. In some embodiments, less than 20% of the population are empty rAAV particles. In some embodiments, less than 25% of the population are empty rAAV particles. In some embodiments, less than 30% of the population are empty rAAV particles. In some embodiments, less than 1%, 2%, 3%, 4%, or 5% of the population are empty rAAV particles.

[0179] In some embodiments, less than 5% of the population are partially filled rAAV particles. In some embodiments, less than 10% of the population are partially filled rAAV particles. In some embodiments, less than 15% of the population are partially filled rAAV particles. In some embodiments, less than 20% of the population are partially filled rAAV particles. In some embodiments, less than 25% of the population are partially filled rAAV particles. In some embodiments, less than 30% of the population are partially filled rAAV particles. In some embodiments, less than 1%, 2%, 3%, 4%, or 5% of the population are partially filled rAAV particles.

[0180] In some embodiments, the rAAV particles in the population comprise a particle selected from the group consisting of AAVhu68, AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc In some embodiments, the rAAV particles in the population comprise capsid proteins of an AAV selected from the group consisting of AAV9, AAV1, and AAVhu68.

[0181] 5.4. Pharmaceutical Compositions

[0182] In another aspect, the present invention provides a pharmaceutical composition comprising rAAV particles prepared by the method disclosed herein. The pharmaceutical composition may comprise a population of the purified rAAV particles and a pharmaceutically acceptable excipient.

[0183] In some embodiments, the rAAV particle comprises a heterologous nucleic acid molecule having a coding sequence for a therapeutic protein. In some embodiments, the heterologous nucleic acid molecule comprises a sequence for gene editing, shRNA, miRNA, or other therapeutic functions. In some embodiments, the heterologous nucleic acid molecule further comprises a regulatory sequence operably linked to a therapeutic gene.

[0184] The pharmaceutical composition can be formulated for intravenous, intramuscular, subcutaneous, intrathecal, intracranial, intracerebroventricular, intradermal, rectal, oral, vaginal, intranasal or inhalation administration. In some embodiments, the pharmaceutical composition is formulated for injection or infusion.

[0185] The pharmaceutical composition can be used to deliver the rAAV to a mammalian subject (eg, a human subject) in need thereof.

[0186] The pharmaceutical composition can be formulated using one or more carriers, excipients, stabilizers, and excipients to, for example: (1) improve stability; (2) increase cell transfection or transduction; (3) allow for sustained or delayed release; (4) alter biodistribution (e.g., targeting the rAAV particles to specific tissues or cell types); (5) increase translation of the encoded protein in vivo; and / or (6) alter the release profile of the encoded protein in vivo.

[0187] Formulations of the pharmaceutical compositions provided herein can include, but are not limited to, saline, which can be formulated with a variety of buffer solutions (e.g., phosphate-buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, water, lipids, liposomes, lipid nanoparticles, polymers, cationic lipid complexes (lipoplexes), core-shell nanoparticles, peptides, proteins, nanoparticle mimetics, and combinations thereof.

[0188] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed hereafter. Generally, such preparative methods include the steps of associating the active ingredient (i.e., rAAV particles) with a carrier and / or one or more other auxiliary ingredients (e.g., excipients, stabilizers, and adjuvants).

[0189] The pharmaceutical composition according to the present disclosure can be prepared, packaged and / or sold in batches as a single unit dose and / or as multiple single unit doses. As used herein, a unit dose refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to a subject and / or a convenient fraction of such dosage, such as half or one-third of such dosage.

[0190] The relative amounts of the active ingredient (i.e., rAAV particles), the pharmaceutically acceptable carrier, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure may vary, depending on the nature, size, and / or condition of the subject being treated and further on the route by which the composition is administered.

[0191] Various carriers, excipients, stabilizers and adjuvants for formulating pharmaceutical compositions and the techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 22nd Revised Edition, Pharmaceutical Press, 2012; incorporated herein by reference in its entirety). Suitable conventional carriers, excipients, stabilizers and adjuvants are also contemplated for use within the scope of the present disclosure.

[0192] 6. Examples

[0193] 6.1. Example 1: AEX purification of rAAV particles

[0194] rAAV particles were purified by a method involving anion exchange chromatography (AEX). Various purification conditions, such as buffer composition, were tested and preferred conditions were selected. Table 1 provides two exemplary conditions (Method A and Method B) tested for the purification of rAAV particles.

[0195]

[0196]

[0197] As summarized in Table 1, in Method A, a feed composition containing a mixture of empty capsids, partial capsids, and complete capsids partially purified by upstream treatments (e.g., depth filtration / diafiltration, TFF concentration, and affinity chromatography) is diluted 50-fold in a loading buffer containing Bis-Tris propane (BTP) buffer (20 mM BTP, 10 mM NaCl, pH 10.2). The same buffer can be used to equilibrate the anion exchange column. This method involves a 2-step load adjustment to pH 10.2. The feed composition is diluted to a BTP buffer containing 0.01% poloxamer 188, pH 10.2.

[0198] Buffer (20 mM) followed by 0.2 M BTP, pH 10.2. In method A, no maximum range was set for the loading capacity (dynamic binding capacity, DBC). The feed composition was applied to an equilibrated anion exchange column (e.g., CIMmultus-QA) at pH 10.2. TM The rAAV particles bound to the chromatography medium were washed with BTP wash buffer (20 mM BTP, 10 mM NaCl, pH 10.2) at 1 CV / min for 10 CV. The bound rAAV particles were eluted with a linear salt gradient to BTP buffer B (20-80% buffer B in 60 CV). UV absorbance was monitored at 260 and 280 nm. Fractions were collected when the A260 / A280 ratio reached an inflection point (≥1) and then stripped with various buffers involving eight steps.

[0199] Method B describes an alternative purification condition for anion exchange chromatography for the purification of rAAV particles. This is a simplified method with fewer steps and reduced buffer usage. Instead of two steps, this method involves a 1-step loading adjustment to pH 8.8 by adding BTP buffer (20 mM) containing 0.01% poloxamer 188, pH 8.9. The loading capacity (dynamic binding capacity, DBC) is limited to approximately 8.5E+13 VG / mL monolith. Similar to method A, the monolith is loaded with an anion exchange column (e.g., CIMmultus-QA TM The rAAV particles bound to the chromatography medium in a column (100 μL) were washed with glycine wash buffer (20 mM glycine, 10 mM NaCl, pH 10.2) at 1 CV / min for 20 CV, followed by a 3-hour retention on the column at pH 10.2. The bound rAAV particles were eluted using glycine buffer B in a linear salt gradient (20-50% buffer B in 30 CV). UV absorbance was monitored at 260 and 280 nm. Fractions were collected when the A260 / A280 ratio reached an inflection point (≥1) and then stripped with a reduced buffer solution involving two steps.

[0200] Method B offers several advantageous features over conventional methods. For example, Method B significantly reduces the number of steps, reduces processing time, reduces the amount of buffers, and reduces the total amount of buffers prepared during the cleaning in place (CIP) and equilibration (CIP / EQ) steps. Note that the CIP / EQ step does not affect processing.

[0201] Method B replaces the BTP buffer with a glycine buffer. This adjustment enhances the buffering capacity of glycine for pH control of the gradient steps (glycine pKa = 9.7, while BTP pKa = 9.0).

[0202] Method A requires a 2-step load adjustment at high pH (pH 10.2). In contrast, Method B uses a 1-step load adjustment to low pH (pH 8.8). Method B lowers the pH to 8.8 or 8.9 to limit the amount of time the product is kept at a high pH (e.g., pH 10.2).

[0203] Method A does not set a maximum range for the loading capacity (dynamic binding capacity (DBC)), while method B limits the loading capacity to approximately 8.5E+13 vg / mL monolith. Typically, the loaded DBC is evaluated at a pH of approximately 8.8. At 8.5E+13 vg / mL, a 10% breakthrough is observed. The loading at production scale is estimated to be 35% (3 x 200 m 2 –400mL CIMQA) and 59% (1x 200m 2–80mL of CIMQA) of DBC.

[0204] During the wash step, Method B replaced BTP with 20 mM glycine, 10 mM NaCl pH 10.2, performed at 1 CV / min for 20 CV, followed by a 3-hour hold on the column. This adjustment increased the amount of intact capsids on the medium at pH 10.2, which increased the yield of intact capsids collected.

[0205] Method B reduces the range of the buffer gradient from 20-80% buffer B in 60CV to 20-50% buffer B in 30CV and additional starting standards, including ≥10 conductivity (mS / cm), >5 milli-absorbance units (mAU) at 280nm and ≥1.0A260 / A280 (inflection point). This adjustment reduces elution time and reduces the time before neutralization adjustment (time under pH 10.2). Ending collection at 50% buffer B provides simplification for production. Additional starting standards (e.g., A260 / A280 ratio) simplification can be used for potential removal and simplification of production.

[0206] During the stripping step, method A involved eight steps using various buffers, while method B involved two steps using reduced buffers. These adjustments reduced processing time, the amount of buffer, and the total number of buffers prepared.

[0207] 6.2. Example 2: Conditions for Purification of rAAV Particles by Anion Exchange Chromatography

[0208] Convective Interactive Media (CIM) QA Monolith (CIMmultus-QA TM , Sartorius) is a strong anion exchange monolith chromatography column containing a cross-linked porous polymethacrylate material with a defined channel size distribution having a diameter exceeding 1000 nm. The monolith chromatography column is used to separate rAAV particles. The rAAV particles processed by upstream treatment (e.g., deep filtration / diafiltration, TFF concentration, and capture chromatography) are diluted 50-fold into Bis-Tris propane (BTP) buffer A (20 mM BTP, 10 mM NaCl, pH 10.2, also referred to as "loading buffer") to form a feed composition. The pH of the feed composition is adjusted to 10.2.

[0209] To determine the effect of loading volume on the percentage of intact rAAV capsids collected, a control experiment was performed using a feed composition containing AAVhu68. The UV absorbance (mAU) spectra at 280 nM and 260 nM are shown in Figure 1A and Figure 1B middle.

[0210] Approximately 1 mL (low volume) of feed composition containing AAVhu68 (including empty capsids, partially filled capsids, and complete capsids) was loaded onto the CIMmultus-QA TM The loading rate was 3.67E+13. The loading rate was 0.67CV / min and the processing rate was 3.13CV / min. At pH 10.2, the loading duration was about 2.23 hours. Figure 1A As shown, these conditions produced approximately 78.2% AAVhu68 particle yield and approximately 70.7% complete AAVhu68 capsids.

[0211] The method was repeated using a total of 4 mL (high volume) of AAVhu68 particles. The loading rate was 3.7E+13. The loading rate was 1 CV / min, and the processing rate was 1 CV / min. The loading duration was approximately 1.25 hours at pH 10.2. Compared to 1 mL (low volume) loaded at a slower rate (0.67 CV / min), these conditions provided an equivalent loading rate, but with a lower amount of time on the column. Figure 1B Approximately 79% AAVhu68 particle yield and approximately 68.5% complete AAVhu68 capsids were shown.

[0212] The data demonstrate that increasing the time on the medium slightly increases the percent yield of particles or the percent of complete capsids collected (as separated from empty and partially filled capsids).

[0213] To determine the effect of high loading ratios on purification efficiency, a feed composition containing AAVhu68 particles (9E+13 Vg / mL, vector genomes per mL) was loaded onto 1 mL CIMmultus-QA at a loading ratio of 2.44E+14 (GC / mL resin) or 3.65E+14 (GC / mL resin). TM On the column. Optionally, the column is pre-balanced with loading buffer. The feed composition is allowed to flow through the column at a loading rate of 0.67 mL / min. The column is washed in wash buffer (20 mM BTP, 20 mM NaCl, pH 10.2) and then eluted with a salt gradient (20-50% gradient) in elution buffer (20 mM BTP, 1 M NaCl, pH 10.2). The volume flow rate is 1.54 CV / min. UV absorbance is monitored at 260 and 280 nm. Fractions are collected when the ratio of A260 / A280 reaches the inflection point (≥1) and then stripped with buffer. The 280 nM and 260 nM UV absorption (mAU) spectra are shown in Figure 2 middle.

[0214] refer to Figure 2, a low loading rate (2.44E+14) produced approximately 81.8% yield and approximately 84.1% complete rAAV capsids, with an elution conductivity of approximately 10.9 ms / cm. The A260 / A280 ratio was greater than one (1.26). In contrast, a high loading rate (3.65E+14) produced approximately 66.33% yield and approximately 83.1% complete rAAV capsids, with an elution conductivity of approximately 11.71 ms / cm. The A260 / A280 ratio was also greater than one (1.37).

[0215] The data show that although high loading rates reduced rAAV particle yield at high pH (pH 10.2), they resulted in comparable percentages of collected intact rAAV capsids. The slow flow rate in the low loading rate experiments likely provided a longer time for rAAV particles to flow through the column when the feed composition was dynamically loaded.

[0216] To determine the effect of loading pH on yield and purity, the supernatant containing rAAV particles was diluted 50-fold in loading buffer as described above, except that magnesium chloride was omitted from the buffer (20 mM BTP, 10 mM NaCl, pH 10), to form a feed composition. The feed composition was adjusted to a pH of 8.8. Approximately 4 mL of the pH 8.8 feed composition was loaded onto the CIMmultus-QA at a nominal loading rate of 3.05E+13. TM The loading rate was about 1 CV / min and the processing rate was about 1 CV / min. The percentage of complete rAAV capsids separated from empty rAAV capsids and partially filled rAAV capsids was about 57.5%, as shown in FIG. Figure 3 shown.

[0217] The data suggest that loading at low pH (eg, pH 8.8) results in a lower percentage of complete rAAV capsids, likely due to overlap of complete and empty peaks (band broadening effect).

[0218] Poloxamer 188 (P188) is a nonionic linear copolymer with an average molecular weight of 8400 Daltons and is also known as PLURONIC F68, FLOCOR and RheothRx. Poloxamer 188 is generally used as a surfactant to stabilize rAAV particles. To determine whether the presence of poloxamer 188 affects the purification of complete rAAV capsids, a feed composition containing rAAV particles (e.g., AAVhu68) was prepared in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2). Poloxamer 188 was not added. The mixture was diluted 50 times for loading. Approximately 1 mL of the feed composition with a pH of 10.2 was loaded onto the CIMmultus-QA TMThe loading rate was 3.03E+13, the loading rate was about 0.67 CV / min, and the processing rate was about 3.13 CV / min. Purification in the absence of poloxamer 188 resulted in a low yield of AAVhu68 particles (about 55.28%) and a low percentage of complete rAAV capsids (about 61%) separated from empty AAVhu68 capsids and partially filled AAVhu68 capsids, as shown in FIG. Figure 4 The data indicate that poloxamer 188 can help improve the yield and purity of rAAV particles.

[0219] To determine the effect of "time on medium" on rAAV particle purification, a feed composition was prepared in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2). The mixture was diluted 20-fold before loading. Approximately 1 mL (load volume) of the feed composition was loaded onto the CIMmultus-QA TM The loading rate was 3.05E+13, the loading speed was about 0.67 CV / min, and the processing speed was about 3.13 CV / min. The loading duration was about 0.85 hours at pH 10.2. Under these conditions, an approximately 82.7% rAAV particle yield was observed, but the percentage of complete rAAV capsids separated from empty rAAV capsids and partially filled rAAV capsids was about 51.8%, as shown in FIG. Figure 5A shown.

[0220] In another experiment, 1 mL of the feed composition diluted 50-fold in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2) was loaded onto the CIMmultus-QA TM The loading rate was 2.44E+14, the loading speed was about 0.67CV / min, and the processing speed was about 3.13CV / min. The loading duration of the 50-fold diluted loaded feed composition was about 14.57 hours, which was an increase of more than 12 hours compared to the 20-fold dilution (higher concentration). Notably, these conditions increased both the yield of rAAV particles (about 81.8%) and the purity of complete rAAV capsids (about 84.1%). The 280nM and 260nM UV absorption (mAU) spectra are shown in Figure 5B middle.

[0221] In another experiment, the loading rate was increased to 3.65E+14, the loading volume (1 mL), loading rate (about 0.67 CV / min), and processing rate (about 3.13 CV / min) remained the same, and the loading duration was about 20.54 hours. Figure 5A The higher loading rate increased the loading duration by approximately 18 hours when compared to the loading duration for the smaller loading volume shown. Figure 5C It was shown that under such conditions, the yield of rAAV was about 66.33%, and the percentage of complete capsids was about 83.1%.

[0222] In this experiment, the time on medium can be decreased by loading a smaller volume, and the time on medium can be increased by loading a larger volume (e.g., Figure 5B The on-column time shown is extended by >12 hours, or as Figure 5C (The time on the column was increased by >18 hours as shown). Overall, the data from the experiments indicate that time on the medium is potentially a decisive factor in improving the isolation of complete rAAV capsids. As time on the medium increases, the yield and purity of complete rAAV particles also increase.

[0223] 6.3. Example 3: Improving Purification of rAAV Particles by Anion Exchange Chromatography by Retention on Resin

[0224] rAAV particles were purified by AEX under conditions where the time on medium was increased by keeping the loaded sample on the medium for a holding duration.

[0225] In the first experiment, the feed composition was prepared in loading buffer (20 mM BTP, 10 mM NaCl, pH 10.2) and diluted 50-fold. TM Before on the post, the feed composition was maintained in a solution at a pH of 10.2 for a duration of 24 hours. After the holding period, 1 mL of the feed composition was loaded onto the post. The load rate was approximately 5.66E+13, and the load rate was approximately 1.54 CV / min, and the treatment rate was approximately 1.54 CV / min. The load duration was approximately 1.49 hours. The post was washed with wash buffer (20 mM BTP, 20 mM NaCl, pH 10.2) for 10 CV with 1 CV / min, and eluted with a salt gradient (20-50% gradient) in elution buffer (20-80% buffer B, 20 mM BTP, 310 mM NaCl, pH 10.2 in 60 CV). UV absorbance was monitored at 260 and 280 nm. The fractions were collected when the ratio of A260 / A280 reached the inflection point (≥1), and then stripped with buffer. Figure 6 The 280 nM and 260 nM UV absorbance (mAU) spectra in ΔΝAAV® showed approximately 60.06% yield and approximately 70.9% complete rAAV capsids. This data indicates that maintaining the solution at a pH of 10.2 did not improve the purity of the complete capsids.

[0226] A second experiment was performed under the same conditions except for the holding step. The loading rate was about 3.0E+13 VG / mL and the loading speed was about 0.67 CV / min. After washing, the rAAV particles were allowed to hold (remain in contact with the medium on the column) at pH 10.2 for a duration (e.g., time on medium) of about 3 hours. Figure 7 As shown, there was about 53.4% yield and about 87% complete rAAV capsids. Figure 6 ), the percentage of complete rAAV capsids was significantly increased by about 16.1% compared to that maintained on medium (about 87%). Figure 8 In addition, compared with Figure 4 As shown, loading at pH 8.8 and eluting at pH 10.2 (approximately 87%) significantly increased the percentage of complete rAAV capsids by approximately 29.5% compared to loading and eluting at pH 8.8 (approximately 57.5%).

[0227] 6.4. Example 4: Increased time on medium is positively correlated with the percentage of complete rAAV capsids

[0228] To determine the effect of time on medium on purification efficiency, the percentage of experimental runs measured by size exclusion chromatography coupled to multi-angle static light scattering (SEC-MALS) (Y-axis) was plotted against time on medium (X-axis). Figure 9A and Figure 9B All variable experiments with comparable conditions were included in the statistical analysis. "Time on medium" refers to the duration of the load at pH 10.2. Although every molecule will not be in contact with the medium on the column for this duration, it is an approximation of the time the first molecule will be in contact with the medium on the column until elution begins. Exceptions are Figure 9B Circled in the middle, where loading occurred at low pH, and increased hold for 3 hours. Figure 9B yes Figure 9A A subset of the data is shown for the time range between 0 and 400 minutes. Figure 9B Prolonged high loads on the column in contact with the medium are excluded, as the effect will not be linear at this point ( Figure 9A The experiments in which the column was loaded at pH 8.8 and kept in contact with the medium at pH 10.2 (180 min) were included ( Figure 9B (circled in ).

[0229] The data show that at pH 10.2, reducing the flow rate increases the time on the medium. Similarly, at pH 10.2, increasing the load volume increases the time on the medium. When including or excluding long-duration loading at pH 10.2 (increased time on the medium at 10.2), the effect is statistically significant (Prob>|t|=0.024, which is less than 0.05). The data illustrate that the time on the medium at pH 10.2 is positively correlated with the percentage of complete rAAV capsids purified and collected.

[0230] 6.5. Example 5: Effect of Buffer Type on Purification of rAAV Particles by Anion Exchange Chromatography

[0231] To determine the effect of buffer type on the percentage of intact rAAV capsids collected, feed compositions were prepared in loading buffer containing BTP (20 mM BTP, 10 mM NaCl, pH 10.2) or in loading buffer containing glycine (20 mM glycine, 10 mM NaCl, pH 10.2). Each mixture was diluted 50-fold and then loaded into 1 mL CIMmultus-QA TM Adjust the load pH to pH 8.8.

[0232] The feed composition in BTP loading buffer was loaded onto the column at a loading pH of 8.8, washed and eluted in BTP buffer at pH 10.2. The loading rate was 3.05E+13. After washing, the rAAV particles were kept in contact with the medium on the column (on-column hold) at pH 10.2 for a duration of 3 hours. The results are shown in Figure 7 middle.

[0233] The feed composition was loaded onto the column in glycine buffer at pH 8.8. The load rate was 3.83E+13. The column was washed with glycine wash buffer (20 mM glycine, 10 mM NaCl, pH 10.2) at 1 CV / min for 20 CV and then kept in contact with the medium on the column at pH 10.2 for 3 hours. The bound rAAV particles were eluted in a linear salt gradient using glycine buffer B (20-50% buffer B in 30 CV). UV absorbance was monitored at 260 and 280 nm. Fractions were collected when the ratio of A260 / A280 reached the inflection point (≥1) and then stripped with buffer. The results are shown in Figure 10 middle.

[0234] Figure 7 and Figure 10The 280 nM and 260 nM UV absorbance (mAU) spectra shown show that loading, washing, maintaining on the column in contact with the medium, and eluting in BTP buffer produced approximately 53.4% yield and approximately 87% complete rAAV capsids. In contrast, loading, washing, maintaining on the column in contact with the medium, and eluting in glycine buffer produced approximately 79.2% yield and approximately 85.3% complete rAAV capsids. This data indicates that the percentage of complete capsids is not affected by the buffer composition, but is affected by maintaining on the column in contact with the medium.

[0235] 6.6. Example 6: Modification of the Anion Exchange Chromatography Method for Purification of rAAV Particles

[0236] This example illustrates modifications to the purification method to improve the efficiency of rAAV particle purification by anion exchange chromatography.

[0237] The feed composition in glycine buffer was diluted 50 times and loaded onto the column at pH 8.8. The loading rate was about 3.83E+13VG / mL and the loading speed was about 0.67CV / min. The column was washed with glycine wash buffer (20mM glycine, 10mM NaCl, pH 10.2) at 1CV / min for 20CV to wash away unbound components. The bound components (e.g., rAAV particles) were allowed to remain on the column at pH 10.2 for a duration of 3 hours. The bound rAAV particles were eluted with glycine buffer B (20-50% buffer B in 30CV) in a linear salt gradient at pH 10.2. Figure 10 As shown, this method produced approximately 79.2% yield and approximately 85.3% complete rAAV capsids. This data indicates that lowering the sample load pH to 8.8, washing with glycine buffer, and holding the column for 3 hours significantly increased the amount of complete rAAV capsids.

[0238] Compared with BTP buffer, which produced approximately 87% complete rAAV capsids ( Figure 7 ) compared to glycine buffer (about 85.3%, Figure 10 ) resulted in a slightly lower percentage of complete capsids, although the difference was not significant. This data suggests that the increase in complete rAAV capsid production is independent of BTP buffer. In addition, the pKa of glycine buffer is higher than that of BTP and provides increased pH control.

[0239] Overall, the data indicate that the dynamic binding capacity is significantly lower at pH 8.8 as determined by the breakthrough at the front of the graph.

[0240] 6.7. Example 7: Improved purification of rAAV particles by anion exchange chromatography independent of AAV serotype

[0241] The rAAV particle purification procedure described in Example 6 was tested with a different AAV serotype, AAV1. A feed composition containing AAV1 was loaded onto the CIMmultus-QA at a loading rate of 2.0E+13 at pH 10.2. TM The column was washed and eluted with BTP buffer at pH 10.2. No hold was performed. Figure 11 showed that this method produced 54.9% yield and 62.1% complete AAV1 capsids.

[0242] The feed composition containing AAV1 was loaded onto the CIMmultus-QA at a loading rate of 4.0E+13 at pH 8.8. TM The column was washed and eluted with glycine buffer at pH 10.2 and allowed to remain in contact with the medium on the column for approximately 3 hours at pH 10.2. Figure 12 The method was shown to produce an 88.7% yield. The A260 / A280 ratio was higher than the control, indicating an increase in the percentage of complete AAV1 capsids.

[0243] A third experiment was performed in which the retention time of the column in contact with the medium was extended to 24 hours. The results of the FPLC analysis are shown in Figure 13A and Figure 13B The A260 / A280 ratio was higher than that of the control, indicating an increase in the percentage of complete AAV1 capsids.

[0244] This data demonstrates that the modified rAAV particle purification method described in Example 6 can be applied to multiple AAV serotypes.

[0245] 6.8 Example 8: Scalable Anion Exchange Chromatography Method for Purification of rAAV Particles

[0246] This example demonstrates that the results of the rAAV particle purification methods described in Examples 6 and 7 can be scaled up for large-scale production and yield high-quality complete rAAV capsids. The following experiments were performed to isolate AAV1 complete capsids.

[0247] The experiment was performed at a slightly adjusted loading pH (pH 8.9 vs. pH 8.8). A feed composition containing AAVhu68 was loaded onto CIMmultus-QA at pH 8.9 at a loading rate of 1.28E+13. TM The column was washed and eluted with glycine buffer at pH 10.2. The rAAV particles bound to the chromatography medium were allowed to remain on the column for approximately 3 hours at pH 10.2. The UV absorption (mAU) spectra at 280 nM and 260 nM are shown in Figure 14The modified method achieved approximately 76.9% yield and approximately 88.3% (by AUC) of complete AAVhu68 capsids. Notably, while minimal traces of partial particles were typically detected in the complete capsid pool collected using conventional methods, no partial particles were detected in the complete capsid pool using the modified method.

[0248] 6.9. Example 9: Application of the Improved Method to Purify rAAV Particles in Different Anion Exchange Chromatography Systems

[0249] This experiment demonstrates the application of modified methods described in Examples 6 and 7 to purify rAAV particles using a different anion exchange chromatography system.

[0250] Capto Q resin (Cytiva), a strong anion exchange resin chromatography column containing a rigid, high-flow agarose matrix modified with a dextran surface extender and quaternary ammonium (Q), was used to purify rAAV particles from upstream purification processes. The loading pH was adjusted for this experiment. The feed composition was loaded at a low pH (pH 8.8) with a loading rate of 9.7E+12, washed with BTP buffer at pH 10.2, and maintained on the column at pH 10.2 for approximately 3 hours. Figure 15 The 280 nM and 260 nM UV absorbance (mAU) spectra in the HPLC-MS / MS spectra indicate that AAV1 particles may be more tightly bound to the chromatography medium, which broadens the peaks and potentially reduces yield. This method achieved approximately 56% yield and approximately 76% intact capsids. This data suggests that additional gradient runs (e.g., extended gradients) may improve yield.

[0251] 7. Equivalents and Incorporation by Reference

[0252] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

[0253] All references, issued patents, and patent applications cited within the text of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method for purifying recombinant adeno-associated virus (rAAV) particles, the method comprising: e) providing a feed composition comprising the rAAV particles, wherein the rAAV particles in the feed composition include empty rAAV particles and complete rAAV particles; f) contacting the feed composition with the chromatography medium under conditions that permit binding of the rAAV particles to the chromatography medium for an on-medium time, wherein the on-medium time is at least 0.5 hours; g) eluting the rAAV particles from the chromatography medium; and h) recovering the purified rAAV particles, thereby enriching for complete rAAV particles. The method according to claim 1 , wherein the time on the medium is longer than 2 hours. The method according to claim 2 , wherein the time on the medium is 2 to 24 hours.

4. The method of claim 2, wherein the time on the medium is longer than 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.

5. The method of any one of claims 1-4, wherein the contacting step comprises maintaining the rAAV particles bound to the chromatography medium in a holding buffer for a holding duration, wherein the holding duration is 0.5 hours or longer. The method according to claim 5 , wherein the holding duration is 0.5 to 24 hours.

7. The method of claim 6, wherein the holding duration does not exceed 3 hours. The method according to claim 7 , wherein the holding duration is 0.5 to 3 hours.

9. The method of claim 8, wherein the holding duration is about 3 hours.

10. The method according to any one of claims 5 to 9, wherein the pH of the holding buffer is between 9.0 and 11.

11. The method of claim 10, wherein the pH of the holding buffer is about pH 10.

2.

12. The method of any one of claims 1-11, wherein the contacting step comprises loading the rAAV particles onto the chromatographic medium for a loading duration.

13. The method of claim 12, wherein the loading duration is longer than 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours or 18 hours.

14. The method according to claim 13, wherein the loading duration is 0.5 to 24 hours.

15. The method according to any one of claims 12 to 14, wherein the load duration and the hold duration total 0.5 to 24 hours.

16. The method of any one of claims 12-15, wherein the load duration and the hold duration total at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours.

17. The method of any one of claims 12-16, wherein the contacting step comprises washing the rAAV particles bound to the chromatography medium with a wash buffer having a pH of 9.0 to 11 after loading the feed composition onto the chromatography medium but before retaining the rAAV particles bound to the chromatography medium.

18. The method of claim 17, wherein the pH of the wash buffer is between pH 9.5 and 10.

5.

19. The method of claim 18, wherein the pH of the wash buffer is about pH 10.

2.

20. The method of any one of claims 17-19, wherein the wash buffer comprises Bis-Tris propane (BTP) or glycine.

21. The method of any one of claims 1-20, wherein the pH of the feed composition is between 8.0 and 8.

9.

22. The method according to any one of claims 1 to 21, wherein the chromatography medium is an anion exchange chromatography medium.

23. The method according to any one of claims 1 to 22, wherein the chromatography medium is an affinity chromatography medium.

24. The method of any one of claims 1 to 23, wherein the elution step is performed with a linear salt gradient.

25. The method of claim 24, wherein the linear salt gradient comprises about 0.001 mM NaCl to about 1000 mM NaCl.

26. The method of claim 24, wherein the linear salt gradient comprises about 0.001 mM NaCl to about 100 mM NaCl.

27. The method of any one of claims 1-23, wherein the elution step is performed with a step salt gradient.

28. The method of claim 27, wherein the step salt gradient comprises about 7 mM NaCl to about 500 mM NaCl.

29. The method of claim 27, wherein the step salt gradient comprises about 70 mM NaCl to about 100 mM NaCl.

30. The method of any one of claims 12-28, wherein the loading step is performed by passing the feed composition through the chromatography medium at a flow rate of 0.1 CV / min to 5 CV / min.

31. The method of any one of claims 1-30, wherein the chromatographic medium comprises one or more amine functional groups.

32. The method of claim 31, wherein the one or more amine functional groups are selected from primary amine, secondary amine, tertiary amine, quaternary amine functional groups, or combinations thereof.

33. The method of claim 31 , wherein the one or more amine functional groups comprise a quaternary amine functional group.

34. The method of claims 31-33, wherein the one or more amine functional groups are bound to a resin, membrane and / or nanofiber chromatography medium.

35. The method of any one of claims 1-34, wherein the chromatographic medium comprises a monolith.

36. The method of any one of claims 1-35, wherein the elution step is performed in a buffer having a pH of 9.5-10.

5.

37. The method of any one of claims 1-36, wherein the rAAV particles comprise a molecule selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAVhu68, AAV.Anc80, AAV. Capsid proteins of AAV such as AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and / or AAV.HSC16.

38. The method of claim 37, wherein the rAAV particles comprise the capsid protein of AAVhu68.

39. The method of claim 37, wherein the rAAV particles comprise capsid proteins of AAV1.

40. The method of claim 37, wherein the rAAV particles comprise capsid protein of AAV9.

41. The method of any one of claims 1-40, further comprising the step of determining the yield of the purified rAAV particles.

42. The method of any one of claims 1-41, wherein the yield of the purified rAAV particles is between 65% and 99%.

43. The method of any one of claims 1-42, further comprising the step of determining the enrichment of whole rAAV particles in the purified rAAV particles.

44. The method of any one of claims 1-43, wherein at least 80% of the purified rAAV particles are whole rAAV particles.

45. The method of claim 44, wherein at least 85% of the purified rAAV particles are whole rAAV particles.

46. The method of any one of claims 1-45, wherein 1% to 40% of the rAAV particles in the feed composition are whole rAAV particles.

47. The method of any one of claims 1-46, wherein the rAAV particles in the feed composition further comprise partially filled rAAV particles.

48. The method of any one of claims 1-47, wherein the chromatography medium is a pre-packed chromatography monolithic column medium.

49. The method of any one of claims 1-47, wherein the chromatography medium is a rigid, high flow rate agarose matrix modified with a dextran surface extender and a strong quaternary ammonium (Q) anion exchanger.

50. The method of any one of claims 1-49, wherein the purified rAAV particles have at least 95% of the potency of the rAAV particles in the feed composition.

51. The method of any one of claims 1-50, wherein the feed composition comprises poloxamer 188.

52. The method of any one of claims 1-51, further comprising a prior step of contacting a sample comprising rAAV particles with an affinity chromatography medium, thereby providing the feed composition.

53. The method according to any one of claims 1-52, further comprising a preceding step of preparing the sample comprising rAAV particles by depth filtration, concentration or diafiltration.

54. The method of claim 53, further comprising a preceding step of preparing the sample comprising rAAV particles by depth filtration, concentration and diafiltration.

55. A population of rAAV particles prepared by the method of any one of claims 1-54.

56. The population of rAAV particles of claim 55, comprising a molecule selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAVhu68, AAV.Anc80, AAV.A Capsid proteins of AAV of nc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 and / or AAV.HSC16.

57. A population of rAAV particles according to claim 56, comprising a capsid protein of an AAV selected from the group consisting of AAV1, AAV9 and AAVhu68.

58. The population of rAAV particles of any one of claims 55-57, wherein at least 70% of the rAAV particles in the population are complete rAAV particles.

59. The population of rAAV particles of claim 58, wherein at least 80% of the rAAV particles in the population are complete rAAV particles.

60. The population of rAAV particles of claim 59, wherein at least 85% or at least 90% of the rAAV particles in the population are complete rAAV particles.

61. A pharmaceutical composition comprising: the rAAV population according to any one of claims 55-60 and a pharmaceutically acceptable excipient.

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